Werner's syndrome, an autosomal recessive disorder characterized by multiple features of accelerated aging, also has a markedly reduced in vitro lifespan. Werner's syndrome fibroblasts exhibit several cell replication abnormalities--including slow growth rates, decreased response to mitogens, chromosomal instability and abnormalities in DNA replication initiation rates. However, the molecular defect responsible for diminished proliferative capacity in these cells in unknown. This proposal aims to identify genes capable of complementing the diminished replicative capacity in Werner's syndrome fibroblasts. A secondary goal is to identify genes able to prolong the lifespan of normal human diploid fibroblasts in vitro. I propose utilizing an amphotropic retrovirus with a selectable genetic drug resistance marker to transfer DNA from normal diploid cells or transformed cells to Werner's syndrome and normal diploid fibroblasts. The basic procedure will involve construction of a cDNA library from young diploid or transformed cells, ligation of the library into a retrovirus vector, packaging the retrovirus, infection of Werner's syndrome or normal fibroblasts, selection of clones with enhanced growth potential and isolation and characterization of complementing genes. Two types of cDNA libraries will be used as sources for complementing genes: one from mRNA isolated from exponentially growing cells, and one from cells in the early S phase of the cell cycle by subtraction hybridization to mRNA derived from Go arrested human diploid cells. After actively proliferating clones are isolated, they will be characterized by determining cumulative doublings before senescence. Retroviarlly inserted genes capable of prolonging lifespan will be identified either by southern hybridization with viral probes or by superinfection with helper virus and rescue of the integrated recombinant retrovirus. I hope to identify the wild type gene which will be used as a probe to isolate the Werner's syndrome defective sequence, and to identify genes that may regulate the lifespan of normal diploid cells.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AG007359-04
Application #
3453157
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Chang, M; Burmer, G C; Sweasy, J et al. (1994) Evidence against DNA polymerase beta as a candidate gene for Werner syndrome. Hum Genet 93:507-12
Chang, M; Tsuchiya, K; Batchelor, R H et al. (1994) Deletion mapping of chromosome 8p in colorectal carcinoma and dysplasia arising in ulcerative colitis, prostatic carcinoma, and malignant fibrous histiocytomas. Am J Pathol 144:1-6
Shedlock, A M; Parker, J D; Crispin, D A et al. (1992) Evolution of the salmonid mitochondrial control region. Mol Phylogenet Evol 1:179-92
Parker, J D; Rabinovitch, P S; Burmer, G C (1991) Targeted gene walking polymerase chain reaction. Nucleic Acids Res 19:3055-60
Burmer, G C; Rabinovitch, P S; Loeb, L A (1991) Frequency and spectrum of c-Ki-ras mutations in human sporadic colon carcinoma, carcinomas arising in ulcerative colitis, and pancreatic adenocarcinoma. Environ Health Perspect 93:27-31
Burmer, G C; Crispin, D A; Kolli, V R et al. (1991) Frequent loss of a p53 allele in carcinomas and their precursors in ulcerative colitis. Cancer Commun 3:167-72
Parker, J D; Burmer, G C (1991) The oligomer extension ""hot blot"": a rapid alternative to Southern blots for analyzing polymerase chain reaction products. Biotechniques 10:94-101
Burmer, G C; Parker, J D; Bates, J et al. (1990) Comparative analysis of human papillomavirus detection by polymerase chain reaction and Virapap/Viratype kits. Am J Clin Pathol 94:554-60
Burmer, G C; Levine, D S; Kulander, B G et al. (1990) c-Ki-ras mutations in chronic ulcerative colitis and sporadic colon carcinoma. Gastroenterology 99:416-20
Burmer, G C; Loeb, L A (1989) Mutations in the KRAS2 oncogene during progressive stages of human colon carcinoma. Proc Natl Acad Sci U S A 86:2403-7

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